CN109633035A - The HPLC finger-print and its construction method of a kind of hawthorn broken wall medicine materical crude slice and application - Google Patents
The HPLC finger-print and its construction method of a kind of hawthorn broken wall medicine materical crude slice and application Download PDFInfo
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Abstract
The present invention relates to Chinese materia medica preparation quality control technology, HPLC finger-print and its construction method and the application of a kind of hawthorn broken wall medicine materical crude slice are specifically disclosed.The construction method includes test solution preparation, reference substance solution preparation, the HPLC analysis determination of chromatographic condition, the measurement of finger-print.The present invention determines its shared peak, obtains standard HPLC finger-print also by the comparison to 10 batch hawthorn broken wall medicine materical crude slice sample HPLC finger-prints.Construction method of the present invention has the advantages that easy to operate, favorable reproducibility, characteristic peak are more, accurate and reliable.HPLC finger-print of the present invention can more fully reflect the characteristic peak information of sample, can carry out quality control to hawthorn broken wall medicine materical crude slice using the HPLC finger-print.
Description
Technical field
The present invention relates to Chinese materia medica preparation quality control technology, and in particular, to a kind of HPLC of hawthorn broken wall medicine materical crude slice
Finger-print and its construction method and application.
Background technique
Hawthorn be rosaceous plant large-fruited Chinese hawthorn Crataegus pimiatificia Bge.var.major N.E.Br. or
The dry mature fruit of hawthorn Crataegus pinnatifida Bge., has food digesting stomach fortifying, and scattered stasis changes turbid lipid-loweringing
Effect is used for meat stagnation, gastral cavilty turgor, diarrhea dysentery abdominal pain, blood stasis closed, postpartum stasis, confidant sting, chest impediment and cardialgia, hernia
Pain, hyperlipidemia, charred FRUCTUS CRATAEGI can enhance promoting digestion effect.
Chinese medicine wall cell disruption medicine materical crude slice is that traditional medicine materical crude slice progress breaking cell wall is ground into granularity point using modern superfine communication technique
Subparticle of the cloth D90 less than 45 μm, then the particle of 30~100 mesh is made.Have color consistent relative to traditional medicine materical crude slice, quality
Uniform, the good feature of stability is the innovative prepared slices of Chinese crude drugs.Since the form that Chinese medicine wall cell disruption medicine materical crude slice does not have traditional Chinese medicine medicine materical crude slice is special
It levies, the dissolution rate of the chemical components such as effective component or index components can be brought to change after broken wall, make identification, the quality of traditional medicine materical crude slice
Detection method cannot be applicable in completely.And according to literature survey, up to 20 are studied for the finger-print of hawthorn at present.Such as Tang Shu
Good equal using Hyperoside as reference peak, establishing with 18 peaks is that shared peak refers to identify the HPLC of Different Provenances fructus crataegi medicinal material
Line atlas calculation；For another example Zhang Liwei etc. is established so that 12 peaks are shared peak using Hyperoside as reference peak and is identified difference
The HPLC fingerprint spectrum method of place of production fructus crataegi medicinal material, after later optimizing method, using 14 peaks as shared peak, for identifying not
With the fructus crataegi medicinal material of concocting method；For another example Chen Baolong etc. is referring to peak with rutin, Hyperoside, and it is shared for establishing with 27 peaks
HPLC fingerprint spectrum method of the haw flavonoids substances at peak, etc..But existing fingerprint map construction method test sample preparation
Time-consuming for operation, and the defects of specificity, stability, poor reproducibility and precision, cannot reflect the defect of prepared slice quality comprehensively,
It there is no the quality determining method for the special hawthorn broken wall medicine materical crude slice of form simultaneously.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of existing hawthorn broken wall prepared slice quality detection method and not
Foot provides HPLC finger-print and its construction method and the application of a kind of hawthorn broken wall medicine materical crude slice.
The object of the present invention is to provide a kind of construction method of the HPLC finger-print of hawthorn broken wall medicine materical crude slice, the construction methods
Have the advantages that easy to operate, favorable reproducibility, characteristic peak are more, accurate and reliable.
Another object of the present invention is to provide a kind of hawthorn broken wall medicine materical crude slice HPLC finger-prints.
Another object of the present invention is to provide a kind of hawthorn broken wall medicine materical crude slice standard HPLC finger-prints.
Another object of the present invention is to provide above-mentioned hawthorn broken wall medicine materical crude slice HPLC finger-print and/or standard HPLC fingerprints
Application of the map in detection hawthorn broken wall prepared slice quality.
Another object of the present invention is to provide a kind of method of quality control of hawthorn broken wall medicine materical crude slice.
To achieve the goals above, the present invention is achieved by following scheme:
A kind of construction method of the HPLC finger-print of hawthorn broken wall medicine materical crude slice, comprising the following steps:
S1. prepared by test solution: precision weighs hawthorn broken wall medicine materical crude slice, is 1:(25~100 by mass volume ratio) it is added
50~100% methanol, in 1.2 ten thousand~1.7 ten thousand turns of 1~5min of homogenized after infiltration, taking-up is cooled to room temperature, with 50~
100% methanol supplies weightlessness, shakes up, and takes filtrate up to test solution through 0.22 μm of filtering with microporous membrane again after coarse filtration；
S2. prepared by reference substance solution: using chlorogenic acid as reference substance, methanol being added to be configured to reference substance solution；
The chromatographic condition of S3.HPLC analysis: crossing octadecylsilane chemically bonded silica chromatographic column for test solution obtained by S1,
20~40 DEG C of column temperature；Gradient elution is carried out by mobile phase of -0.1% formic acid solution of acetonitrile-methanol；Flow velocity is 0.6~1.2mL/
min；Detection wavelength is 280nm；
Mobile phase acetonitrile volume fraction during gradient elution changes are as follows: 0~10min, 5%~8%；10min
~20min, 8%~8%；20min~30min, 8%~11%；30min~45min, 11%~14%；45min~70min,
14%~28%；70min~75min, 28%~5%；75min~85min, 5%~5%；
0.1% formic acid solution of mobile phase volume fraction during gradient elution changes are as follows: 0~10min, 95%~
90%；10min~20min, 90%~90%；20min~30min, 90%~87%；30min~45min, 87%~84%；
45min~70min, 84%~70%；70min~75min, 70%~95%；75min~85min, 95%~95%；
S4. test solution is analyzed under the chromatographic condition of step S3 to get the HPLC finger-print of hawthorn broken wall medicine materical crude slice.
It is time-consuming shorter in order to keep operation more easy in the method for building up of finger-print, and make each in finger-print
Characteristic peak has preferable separating degree, peak shape, while needing the detection time of reasonable control finger-print, for test sample
The selection of preparation method and condition of gradient elution is most important, preparation method and ladder of the those skilled in the art in selection test sample
It requires to pay a large amount of experiment when spending elution requirement, constantly the preparation method of test sample and condition of gradient elution is carried out
Optimization, because chemical component therein is multifarious for different Chinese medicine and preparation, the extraction of different medicinal materials and preparation
The elution requirement of condition and finger-print has no reference value, and the selection of test sample preparation condition not only affects finger-print
Peak number, while being typically also the step taken a long time in entire finger-print operating process, and mobile phase ratio
Minor change very big influence will be caused to finger-print, will such as will affect the separating degree and peak shape of characteristic peak.
Present inventor through a large number of experiments, gropes preparation method of test article and condition of gradient elution,
Above-mentioned preparation method of test article and condition of gradient elution has finally been determined.As the present invention has investigated the extraction side different to test sample
The investigation of method and the different extraction conditions using homogenization process, while many different mobile phases have also been investigated to finger-print
Influence, the results showed that, using above-mentioned preparation method of test article and above-mentioned mobile phase, in finger-print the information at gained peak compared with
It is more；Baseline is more steady, and separating degree, peak shape and column effect are best.Different Detection wavelengths, column temperature have also been investigated to the shadow of finger-print
It rings, the finger-print as a result obtained under above-mentioned wavelength and column temperature, ingredient is relatively more, each peak height ratio is moderate, baseline ratio
It is relatively steady.
To sum up, construction method of the present invention can quickly and accurately identify the quality good or not of product, have it is easy to operate,
Time-consuming short, stable, precision height, high repeatability and other advantages.The hawthorn broken wall medicine materical crude slice HPLC detected using the construction method is referred to
Line map chemical component peak is relatively more, each characteristic peak height ratio is moderate, and baseline is more steady, and separating degree, peak shape and column are imitated.
Preferably, the mass volume ratio of hawthorn broken wall medicine materical crude slice described in the step S1 and methanol is 1:25.
Preferably, the volume fraction of methanol described in step S1 is 70%.
Preferably, the condition of homogenized described in step S1 is 1.2 ten thousand turns, and the time of processing is 2min.
Preferably, reference substance solution described in step S2 is prepared by following steps: precision weighs chlorogenic acid pair
According to product, methanol is added to be configured to the reference substance solution that concentration is 0.5mg/mL.
Preferably, the column temperature of chromatographic condition described in step S3 is 30 DEG C, and the flow velocity for carrying out gradient elution is 0.7mL/
min。
Preferably, when carrying out chromatography, the test solution sample volume is 20 μ L, and reference substance solution sample volume is 10
μL。
A kind of hawthorn broken wall medicine materical crude slice HPLC finger-print is also claimed in the present invention, is constructed by above-mentioned construction method
It arrives.
A kind of hawthorn broken wall medicine materical crude slice standard HPLC finger-print is also claimed in the present invention, be by above-mentioned construction method,
Building obtains the HPLC finger-print of 10 batch hawthorn broken wall medicine materical crude slice samples, and obtained HPLC finger-print is imported finger-print
It analyzes software, generates finger-print common pattern to obtain the final product；The standard HPLC finger-print has 11 shared peaks.With No. 6 peaks
Retention time is reference, the relative retention time at remaining 10 shared peak is respectively 0.278,0.295,0.393,0.746,
0.888、1.130、1.180、1.423、1.438、1.456。
The hawthorn broken wall medicine materical crude slice HPLC finger-print measured by above-mentioned construction method, can more fully reflect the spy of sample
Peak information is levied, quality control can be carried out to hawthorn broken wall medicine materical crude slice using the HPLC finger-print.Therefore, above-mentioned hawthorn broken wall drink
The application of piece HPLC finger-print and/or standard HPLC finger-print in detection hawthorn broken wall prepared slice quality is also protected in the present invention
Within the scope of shield.
A kind of method of quality control of hawthorn broken wall medicine materical crude slice is also claimed in the present invention, includes the following steps: to take hawthorn broken
Wall medicine materical crude slice measuring samples, according to the HPLC finger-print of above-mentioned construction method building measuring samples；Then with above-mentioned hawthorn broken wall
Medicine materical crude slice standard HPLC finger-print is compared；If in the HPLC finger-print of measuring samples, 11 character pair peaks are presented, and
It is calculated by similarity evaluation, the HPLC finger-print and standard HPLC finger-print of measuring samples
Similarity be not less than 0.85, be referring to peak with No. 6 peaks, remaining each peak relative retention time then judges in the range of ± 5%
For qualified samples.
Compared with prior art, the invention has the following advantages:
(1) construction method provided by the present invention have the time-consuming short, favorable reproducibility of easy to operate, test solution preparation,
More, the accurate and reliable advantage of characteristic peak can be mentioned with the quality of overall monitor hawthorn broken wall medicine materical crude slice product for pharmaceutical production and detection
The high standard for having supplied quality to control guarantees that drug uses safe and effective to ensure the stability of product quality.
(2) the hawthorn broken wall medicine materical crude slice HPLC finger-print chemical component peak obtained using construction method of the invention is relatively
More, each characteristic peak height ratio is moderate, and baseline is more steady, and separating degree, peak shape and column are imitated, and can more fully reflect sample
Characteristic peak information can be applied to the quality control of hawthorn broken wall medicine materical crude slice product.
Detailed description of the invention
Fig. 1 is the HPLC finger-print of chlorogenic acid reference substance.Wherein, No. 4 peaks are chlorogenic acid.
Fig. 2 is the HPLC finger-print of 10 batches of hawthorn broken wall medicine materical crude slice.
Fig. 3 is the standard HPLC finger-print of hawthorn broken wall medicine materical crude slice.
Fig. 4 is that hawthorn broken wall medicine materical crude slice HPLC fingerprint analysis method specificity investigates chromatogram.
Fig. 5 is hawthorn broken wall medicine materical crude slice HPLC finger-print Precision Analyze map.
Fig. 6 is that hawthorn broken wall medicine materical crude slice HPLC finger-print repeatability analyzes map.
Fig. 7 is hawthorn broken wall medicine materical crude slice HPLC finger-print stability analysis map.
Fig. 8 is hawthorn broken wall medicine materical crude slice finger-print difference chromatogram column analysis map.
Fig. 9 is that hawthorn broken wall medicine materical crude slice finger-print difference instrument analyzes map.
Figure 10 is that hawthorn broken wall medicine materical crude slice test sample prepares Extraction solvent investigation chromatogram.
Figure 11 is that hawthorn broken wall medicine materical crude slice test sample prepares Extraction solvent concentration investigation chromatogram.
Figure 12 is that hawthorn broken wall medicine materical crude slice test sample prepares extracting mode investigation chromatogram.
Figure 13 is that hawthorn broken wall medicine materical crude slice test sample preparation homogenate revolving speed investigates chromatogram.
Figure 14 is hawthorn broken wall medicine materical crude slice test sample preparation homogenate extraction time to investigate chromatogram.
Figure 15 is that hawthorn broken wall medicine materical crude slice test sample solid-liquid ratio investigates chromatogram.
Figure 16 is that hawthorn broken wall medicine materical crude slice fingerprint map analyzing flow phase system investigates chromatogram.Wherein, S1 is methanol-
0.1% acetic acid, S2 are -0.1% formic acid of methanol, and S3 is -0.1% phosphoric acid of methanol, and S4 is -0.1% acetic acid of methanol, and S5 is methanol -
Water, S6 are -0.1% acetic acid of acetonitrile, and S7 is -0.1% formic acid of acetonitrile, and S8 is -0.1% phosphoric acid of acetonitrile, and S9 is -0.1% second of acetonitrile
Acid, S10 are acetonitrile-water, and S11 is -0.1% formic acid of acetonitrile-methanol.
Figure 17 is that hawthorn broken wall medicine materical crude slice fingerprint map analyzing eluent gradient optimizes chromatogram；Wherein A is gradient 1-6, and B is
Gradient 7-11.
Figure 18 is that hawthorn broken wall medicine materical crude slice fingerprint map analyzing flow velocity investigates chromatogram.
Figure 19 is that hawthorn broken wall medicine materical crude slice fingerprint map analyzing difference column temperature investigates chromatogram.
Figure 20 is that hawthorn broken wall medicine materical crude slice fingerprint map analyzing sampling volume investigates chromatogram.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment is made the present invention and is further elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
1, instrument
Electric-heated thermostatic water bath HWS 24 (Shanghai one permanent Science and Technology Ltd.), electronic balance ME204 (a ten thousandth,
METTLER TOLEOD), electronic balance MS105 (ten a ten thousandths, METTLER TOLEOD), high performance liquid chromatograph (An Jie
Human relations 1200RRLC, Agilent Technologies (China) Co., Ltd), high performance liquid chromatograph (Waters 2695/2988, water generation section
Skill (Shanghai) Co., Ltd.), high performance liquid chromatograph (Thermo U3000, Thermo Fischer Scient Inc.), numerical control supersonic
(bright and limpid D24UV type, U.S. Milipore are public for washer (KQ-700DE type, Kunshan Ultrasonic Instruments Co., Ltd.), ultrapure water machine
Department), adjustable high speed refiner (Changzhou FSH-2A Pu Tian instrument manufacturing Co., Ltd), Phenomenex C18 (5 μm, 250mm ×
4.6mm), Agilent Zorbax SB-Aq C18 (5 μm, 250mm × 4.6mm), Shiseido PAK C18 (5 μm, 250mm
× 4.6mm), YMC-Triart C18 (5 μm, 250mm × 4.6mm).
2, reagent
(chromatographically pure, connection is extraordinary molten when Anhui for acetonitrile (chromatographically pure, Anhui Fulltime Specialized Solvent & Reagent Co., Ltd.), methanol
Agent limited liability company), phosphoric acid (Guangzhou Chemical Reagent Factory), formic acid (Guangzhou Chemical Reagent Factory), acetic acid (Guangzhou chemical reagent
Factory), glacial acetic acid (Guangzhou Chemical Reagent Factory), methanol (analyze pure, Guangzhou Chemical Reagent Factory), redistilled water (self-control).
3, sample
Hawthorn broken wall medicine materical crude slice UGP20180505, UGP20181201, UGP20181202, UGP20181203,
UGP20181204、UGP20181205、UGP20181206、UGP20181207、UGP20181208、UGP20181209、
UGP20181210 totally 11 batches of samples, are provided by Zhongshan City Zhongzhi Traditional Chinese Medicine Decoction Pieces Co., Ltd.
4, reference substance
Chlorogenic acid (is purchased from National Institute for Food and Drugs Control, 110753-201405).
Data processing of the present invention uses Chinese Pharmacopoeia Commission " similarity evaluation " (2012
Version) carry out chromatography similarity evaluation.
The construction method of the HPLC finger-print of 1 hawthorn broken wall medicine materical crude slice of embodiment
A kind of construction method of the HPLC finger-print of hawthorn broken wall medicine materical crude slice, comprising the following steps:
S1. prepared by test solution: precision weighs hawthorn broken wall medicine materical crude slice, is that 70% first is added in 1:25 by mass volume ratio
Alcohol, in 1.2 ten thousand turns of homogenized 2min after infiltration, taking-up is cooled to room temperature, and is supplied weightlessness with 70% methanol, is shaken up, after coarse filtration
Again through 0.22 μm of filtering with microporous membrane, take filtrate up to test solution；
S2. prepared by reference substance solution: using chlorogenic acid as reference substance, methanol being added to be configured to reference substance solution；
The chromatographic condition of S3.HPLC analysis: crossing octadecylsilane chemically bonded silica chromatographic column for test solution obtained by S1,
30 DEG C of column temperature；Gradient elution is carried out by mobile phase of -0.1% formic acid solution of acetonitrile-methanol；Flow velocity is 0.7mL/min；Test sample
Solution sample volume is 20 μ L, and reference substance solution sample volume is 10 μ L；Detection wavelength is 280nm；
Mobile phase acetonitrile volume fraction during gradient elution changes are as follows: 0~10min, 5%~8%；10min
~20min, 8%~8%；20min~30min, 8%~11%；30min~45min, 11%~14%；45min~70min,
14%~28%；70min~75min, 28%~5%；75min~85min, 5%~5%；
0.1% formic acid solution of mobile phase volume fraction during gradient elution changes are as follows: 0~10min, 95%~
90%；10min~20min, 90%~90%；20min~30min, 90%~87%；30min~45min, 87%~84%；
45min~70min, 84%~70%；70min~75min, 70%~95%；75min~85min, 95%~95%；
S4. test solution is analyzed under the chromatographic condition of step S3 to get the HPLC finger-print of hawthorn broken wall medicine materical crude slice.
The foundation of 2 hawthorn broken wall medicine materical crude slice finger-print common pattern of embodiment
1, pointing out referring to peak
According to the construction method of embodiment 1, the HPLC finger-print of reference substance solution chlorogenic acid is measured, records chromatogram,
And be compared it with hawthorn broken wall medicine materical crude slice HPLC finger-print, analyze the retention time and UV absorption feelings at corresponding peak
Condition, as shown in Figure 1.
Identify: No. 4 peaks are chlorogenic acid.Wherein No. 6 peak heights are moderate, and separating degree is good, and retention time is moderate, are selected as ginseng
According to peak.
2, the foundation of finger-print and the confirmation at shared peak
Take 10 batch hawthorn broken wall medicine materical crude slice (lot number UGP20181201, UGP20181202, UGP20181203,
UGP20181204、UGP20181205、UGP20181206、UGP20181207、UGP20181208、UGP20181209、
UGP20181210), test solution is prepared according to the construction method of embodiment 1, and measures 10 batches of test solutions, record color
Spectrogram.Map is opened to gained 10 using similarity evaluation and carries out similarity analysis, it is determined that 11
A chromatographic peak is that hawthorn broken wall medicine materical crude slice shares fingerprint peaks, sees Fig. 2 and Fig. 3, the similarity of 10 batches of samples is 0.9 or more, as a result
It is shown in Table 1.
Wherein, No. 6 peak heights are moderate, and separating degree is good, and retention time is moderate, can be used as referring to peak.Remaining 10 shared color
Spectral peak is respectively as follows: 0.278 with the average relative retention time referring to peak, 0.295,0.393,0.746,0.888,1.130,
1.180,1.423,1.438,1.456, it the results are shown in Table 2.
1 hawthorn broken wall medicine materical crude slice fingerprint similarity of table
2 hawthorn broken wall medicine materical crude slice finger-print of table shares the relative retention time at peak
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | |
| UGP20181201 | 0.278 | 0.295 | 0.393 | 0.747 | 0.889 | 1.000 | 1.130 | 1.179 | 1.422 | 1.437 | 1.455 |
| UGP20181202 | 0.278 | 0.295 | 0.392 | 0.747 | 0.888 | 1.000 | 1.129 | 1.179 | 1.422 | 1.437 | 1.454 |
| UGP20181203 | 0.278 | 0.295 | 0.393 | 0.748 | 0.889 | 1.000 | 1.129 | 1.178 | 1.421 | 1.436 | 1.454 |
| UGP20181204 | 0.278 | 0.295 | 0.393 | 0.747 | 0.888 | 1.000 | 1.129 | 1.179 | 1.423 | 1.438 | 1.456 |
| UGP20181205 | 0.278 | 0.295 | 0.393 | 0.746 | 0.888 | 1.000 | 1.131 | 1.180 | 1.423 | 1.438 | 1.456 |
| UGP20181206 | 0.278 | 0.295 | 0.393 | 0.746 | 0.887 | 1.000 | 1.131 | 1.181 | 1.424 | 1.439 | 1.457 |
| UGP20181207 | 0.277 | 0.295 | 0.392 | 0.745 | 0.887 | 1.000 | 1.131 | 1.180 | 1.423 | 1.439 | 1.456 |
| UGP20181208 | 0.278 | 0.296 | 0.393 | 0.747 | 0.888 | 1.000 | 1.131 | 1.180 | 1.423 | 1.438 | 1.456 |
| UGP20181209 | 0.278 | 0.295 | 0.393 | 0.746 | 0.887 | 1.000 | 1.131 | 1.180 | 1.423 | 1.438 | 1.456 |
| Opposite Average residence time | 0.278 | 0.295 | 0.393 | 0.746 | 0.888 | 1.000 | 1.130 | 1.180 | 1.423 | 1.438 | 1.456 |
| Opposite Average residence time RSD value | 0.101 | 0.084 | 0.073 | 0.122 | 0.057 | 0.000 | 0.079 | 0.090 | 0.063 | 0.059 | 0.069 |
3 methodology validation of embodiment
1, specificity is investigated
Taking lot number is the hawthorn broken wall medicine materical crude slice of UGP20180505, and it is molten to prepare test sample according to the method for 1 step S1 of embodiment
Liquid and blank test solution, sample introduction measure according to the chromatographic condition of 1 step S3 of embodiment, record chromatogram, as shown in Figure 4.
Solvent influences the chromatogram of sample smaller substantially without absorption under chromatographic condition as the result is shown.Show that the method established is exclusive
Property is good.
2, instrument precision
Taking lot number is the hawthorn broken wall medicine materical crude slice of UGP20180505, and it is molten to prepare test sample according to the method for 1 step S1 of embodiment
Liquid continuous sample introduction 6 times, is measured according to the chromatographic condition of 1 step S3 of embodiment, is recorded chromatogram (see Fig. 5).Using Chinese medicine chromatography
Fingerprint similarity evaluation system carries out full spectrum similarity analysis to 6 parts of maps of gained, and similarity analysis the results are shown in Table 3, phase
Like degree up to 1.000.Using No. 6 peaks as reference, the relative retention time RSD value at remaining peak is respectively less than 1.0%, relative peak area
RSD value is less than 3.0%.It investigates the result shows that instrument precision meets the requirements.
3 hawthorn broken wall medicine materical crude slice fingerprint precision similarity analysis of table
| Spectrogram | 1 | 2 | 3 | 4 | 5 | 6 |
| 1 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 2 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 3 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 4 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 5 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| 6 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
3, repetitive test
Taking lot number is the hawthorn broken wall medicine materical crude slice of UGP20180505, and it is molten to prepare test sample according to the method for 1 step S1 of embodiment
Liquid repeats to prepare 6 samples, measures, records chromatogram according to the chromatographic condition of 1 step S3 of embodiment (see Fig. 6).Using Chinese medicine
Chromatographic fingerprinting similarity evaluation system carries out full spectrum similarity analysis to 6 parts of maps of gained, and similarity analysis the results are shown in Table
4, similarity is up to 0.997 or more.Using No. 6 peaks as reference, the relative retention time RSD value at remaining peak is respectively less than 1.0%, relatively
Peak area RSD value is less than 3.0%.It investigates the result shows that the repeatability of method meets the requirements.
4 hawthorn broken wall medicine materical crude slice finger-print repeatability similarity analysis of table
| Spectrogram | 1 | 2 | 3 | 4 | 5 | 6 |
| 1 | 1.000 | 0.999 | 1.000 | 0.999 | 0.998 | 0.997 |
| 2 | 0.999 | 1.000 | 0.999 | 0.998 | 0.998 | 0.998 |
| 3 | 1.000 | 0.999 | 1.000 | 0.999 | 0.998 | 0.998 |
| 4 | 0.999 | 0.998 | 0.999 | 1.000 | 0.999 | 0.999 |
| 5 | 0.998 | 0.998 | 0.998 | 0.999 | 1.000 | 1.000 |
| 6 | 0.997 | 0.998 | 0.998 | 0.999 | 1.000 | 1.000 |
4, serviceability test
(1) stability test of test solution
Taking lot number is the hawthorn broken wall medicine materical crude slice of UGP20180505, and it is molten to prepare test sample according to the method for 1 step S1 of embodiment
Liquid is measured respectively at 0,2,4,8,12,24 hour sample introduction according to the chromatographic condition of 1 step S3 of embodiment, record chromatogram (see
Fig. 7).Full spectrum similarity analysis, similarity are carried out to 6 parts of maps of gained using similarity evaluation
Analysis the results are shown in Table 5, and similarity is up to 0.998 or more.Using No. 6 peaks as reference, the relative retention time RSD value at remaining peak is small
In 1.0%, relative peak area RSD value is less than 3.0%.It investigates the result shows that instrument precision meets the requirements.Investigate the result shows that
The stability of method meets the requirements.
5 hawthorn broken wall medicine materical crude slice finger-print stability similarity analysis of table
| Spectrogram | 0h | 2h | 4h | 8h | 12h | 24h |
| 0h | 1.000 | 1.000 | 1.000 | 0.998 | 0.999 | 1.000 |
| 2h | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 1.000 |
| 4h | 1.000 | 1.000 | 1.000 | 0.998 | 0.998 | 1.000 |
| 8h | 0.998 | 0.999 | 0.998 | 1.000 | 1.000 | 0.998 |
| 12h | 0.999 | 0.999 | 0.998 | 1.000 | 1.000 | 0.998 |
| 24h | 1.000 | 1.000 | 1.000 | 0.998 | 0.998 | 1.000 |
(2) different chromatographic columns compare
Taking lot number is the hawthorn broken wall medicine materical crude slice of UGP20180505, and it is molten to prepare test sample according to the method for 1 step S1 of embodiment
Phenomenex C18 (5 μm, 250mm × 4.6mm), YMC-Triart C is respectively adopted in liquid18(5 μm, 250mm × 4.6mm),
Agilent Zorbax SB-Aq C18 (5 μm, 250mm × 4.6mm) and Shiseido PAK C18 (5 μm, 250mm ×
4.6mm) four different chromatographic columns are measured according to the chromatographic condition of 1 step S3 of embodiment, are recorded chromatogram (see Fig. 8), use
Similarity evaluation carries out full spectrum similarity analysis, similarity analysis result to 4 parts of maps of gained
6 are shown in Table, similarity is 0.986 or more.Show that this method applies generally to different chromatographic columns.
6 hawthorn broken wall medicine materical crude slice finger-print difference chromatographic column similarity analysis of table
(3) different instruments compare
Taking lot number is the hawthorn broken wall medicine materical crude slice of UGP20180505, and it is molten to prepare test sample according to the method for 1 step S1 of embodiment
Liquid, respectively on Waters 2695/2998 and Thermo U3000 liquid chromatograph using Shiseido PAK C18 (5 μm,
250mm × 4.6mm) chromatographic column, it measures, records chromatogram (see Fig. 9), according to the chromatographic condition of 1 step S3 of embodiment
Medicine chromatographic fingerprinting similarity evaluation system carries out full spectrum similarity analysis to 3 parts of maps of gained, and similarity is full up to 0.999
Sufficient methodology requirement.
7 hawthorn broken wall medicine materical crude slice finger-print difference instrument similarity analysis of table
To sum up, methodological study the result shows that, the hawthorn broken wall medicine materical crude slice HPLC fingerprint analysis method established can expire
Sufficient methodological study requirement, and it is easy to operate, it can be applied to the HPLC fingerprint map analyzing of hawthorn broken wall medicine materical crude slice.
The optimization of 4 sample solution preparation method of embodiment
Under conditions of chromatographic condition is fixed, optimize the preparation method of test solution.Fixed chromatographic condition is as follows:
Chromatographic column: Shiseido PAK C18 (5 μm, 250mm × 4.6mm)；
Mobile phase: -0.01% aqueous formic acid of acetonitrile；Gradient elution: 0min-20min-55min-75min-80min-
90min, acetonitrile change 5%-10%-14%-17%-5%-5%；
Flow velocity: 1mL/min；Column temperature: 30 DEG C；Detection wavelength: 280nm；10 μ L of sample injection volume；10 μ of reference substance sample volume
L。
1, the investigation of Extraction solvent
Influence of the 2 kinds of solvents of ethyl alcohol and methanol to hawthorn broken wall medicine materical crude slice extraction efficiency is investigated, specific step is as follows.
Sample treatment: taking hawthorn broken wall medicine materical crude slice 2.0g, accurately weighed 2 parts, set in stuffed conical flask, and a copy of it is added
70% ethyl alcohol 50mL, in addition a to be added 70% methanol 50mL, weighed weight, ultrasonic extraction 30min is let cool after taking-up, with mentioning
Solvent is taken to mend weight, coarse filtration, 0.22 μm of filtering with microporous membrane takes filtrate to get test sample.
Using the chromatographic condition of above-mentioned fixation, above-mentioned 2 parts of samples are analyzed, compares different solvents and extracts difference.With sample spectra
Figure appearance number is more, peak is evenly distributed and a height of selection criteria of chlorogenic acid content, filters out optimum extraction solvent.As a result see figure
10。
From the above results, the chromatogram that the chromatogram that 70% methanol extracts is extracted with 70% ethyl alcohol is no different, main peak kind
Class is almost the same, and similarity is higher, but the content of the chlorogenic acid of 70% methanol extraction is slightly above the extraction of 70% ethyl alcohol.In summary
Data select methanol as the Extraction solvent of hawthorn test sample.
2, the investigation of methanol extracting concentration
It is broken to hawthorn that 50% methanol, 70% methanol, 80% methanol, the methanol solution of 4 kinds of various concentrations of methanol are investigated respectively
The influence of wall medicine materical crude slice sample chemical constituents extraction, specific step is as follows.
Sample treatment: taking hawthorn broken wall medicine materical crude slice 2.0g, accurately weighed 4 parts, set in stuffed conical flask, No. 1 addition
50mL50% methanol, No. 2 addition 70% methanol of 50mL, No. 3 addition 80% methanol of 50mL, No. 4 addition 50mL methanol are weighed heavy
Amount, ultrasonic extraction 30min take out, are cooled to room temperature, supply weightlessness with Extraction solvent, shake up, 0.22 μm of filtering with microporous membrane,
Take filtrate to get test sample.
Using the chromatographic condition of above-mentioned fixation, above-mentioned 4 parts of samples are analyzed, compares different solvents and extracts difference.With sample spectra
Figure appearance number is more, peak is evenly distributed and with a height of selection criteria of chlorogenic acid content, filters out optimum extraction solvent.As a result see figure
11。
As can be seen from the results, the sample chromatogram figure appearance number that 4 kinds of concentration methanol extracts is no different substantially, main peak type one
It causes, 70% methanol of response at principal component peak extracts slightly higher.Considering from solvent cost is saved, the methanol for screening out high concentration extracts,
The final Extraction solvent for choosing 70% methanol as hawthorn broken wall medicine materical crude slice.
3, the investigation of extracting mode
(1) ultrasonic extraction: taking hawthorn broken wall medicine materical crude slice 2.0g, and sample is placed in stuffed conical flask, and 70% first of 50mL is added
Alcohol, weighed weight, ultrasonic extraction 40min take out, are cooled to room temperature, supply weightlessness with Extraction solvent, shake up, pass through again after coarse filtration
0.22 μm of filtering with microporous membrane, takes filtrate.
(2) heating water bath refluxing extraction: taking hawthorn broken wall medicine materical crude slice 2.0g, and sample is placed in stuffed conical flask, is added
50mL70% methanol, weighed weight, heating water bath reflux 30min take out, supply weightlessness after cooling, shake up, pass through again after coarse filtration
0.22 μm of miillpore filter filtration, takes filtrate.
(3) homogenate is extracted: taking hawthorn broken wall medicine materical crude slice 2.0g, sample is placed in stuffed conical flask, and 70% first of 50mL is added
Alcohol, weighed weight, 1.2 ten thousand turns of homogenate of revolving speed extract 5min, take out, be cooled to room temperature, supply weightlessness with Extraction solvent, shake up,
It is filtered again through 0.22 μm of miillpore filter after coarse filtration, takes filtrate.
(4) Soxhlet extraction: taking hawthorn broken wall medicine materical crude slice 2.0g, and sample is placed in stuffed conical flask, and 70% first of 50mL is added
Alcohol, weighed weight, heating water bath reflux 30min take out, supply weightlessness after cooling, shake up, filter again through 0.22 μm of micropore after coarse filtration
Membrane filtration mistake, takes filtrate.
Using the chromatographic condition of above-mentioned fixation, above-mentioned 4 parts of samples are analyzed, the extraction effect of Different Extraction Method is compared.With
Sample spectrogram appearance number is more, peak is evenly distributed and a height of selection criteria of chlorogenic acid equal size, screens extracting mode.As a result as schemed
12。
As can be seen from the results, sample chromatogram figure appearance number obtained by 4 kinds of extracting modes is no different substantially, and main peak type is consistent,
The response at principal component peak also no significant difference.In view of homogenate extraction is time-consuming short, easy, therefore homogenate is selected to extract as final
Extracting mode.
4, the investigation of revolving speed is extracted
It investigates different rotating speeds and extracts (1.2 ten thousand turns, 1.3 ten thousand turns, 1.4 ten thousand turns, 1.5 ten thousand turns, 1.7 ten thousand turns) to hawthorn broken wall drink
The influence of piece extraction effect, specific step is as follows.
Accurately weighed 5 portions of hawthorn broken wall medicine materical crude slice is taken, every part of 2g is set in stuffed conical flask, is separately added into 70% first of 50mL
Alcohol, weighed weight, No. 1 1.2 ten thousand turns of extraction 5min, No. 2 1.3 ten thousand turns of extraction 5min, No. 3 1.4 ten thousand turns of extraction 5min, No. 4 1.5 ten thousand
Turn to extract 5min, No. 5 1.7 ten thousand turns of extraction 5min, takes out, be cooled to room temperature, supply weightlessness with 70% methanol, shake up, after coarse filtration
Again through 0.22 μm of filtering with microporous membrane, filtrate is taken.
Using the chromatographic condition of above-mentioned fixation, above-mentioned 5 parts of samples are analyzed, the extraction effect of Different Extraction Method is compared.With
Sample spectrogram appearance number is more, peak is evenly distributed and a height of selection criteria of chlorogenic acid content, and revolving speed is extracted in screening.As a result as schemed
13。
As can be seen from the results, influence of the revolving speed to extraction effect is unobvious.Final choice revolving speed 1.2 ten thousand transfer to for test sample it is molten
The extraction revolving speed of liquid preparation.
5, the investigation of extraction time
Different homogenate extraction times (1min, 2min, 3min, 4min, 5min) are investigated to hawthorn broken wall medicine materical crude slice extraction effect
Influence, specific step is as follows.
Accurately weighed 5 parts of hawthorn broken wall medicine materical crude slice are taken, every part of 2g is set in stuffed conical flask, is separately added into 70% first of 50mL
1min is extracted in alcohol, weighed weight, No. 1 1.2 ten thousand turns of homogenate, and 2min is extracted in No. 2 1.2 ten thousand turns of homogenate, and No. 3 1.2 ten thousand turns of homogenate are extracted
4min is extracted in 3min, No. 4 1.2 ten thousand turns of homogenate, and No. 5 1.2 ten thousand turns of homogenate extract 5min, take out, be cooled to room temperature, with 70% methanol
Weightlessness is supplied, is shaken up, takes filtrate through 0.22 μm of filtering with microporous membrane again after coarse filtration.
Using the chromatographic condition of above-mentioned fixation, above-mentioned 5 parts of samples are analyzed, the extraction effect of Different Extraction Method is compared.With
Sample spectrogram appearance number is more, peak is evenly distributed and a height of selection criteria of chlorogenic acid equal size, screens extraction time.As a result as schemed
14。
As can be seen from the results, influence of the Homogenization time to extraction effect is unobvious.But with the extension of time, extraction efficiency has
(peak area at principal component peak slightly improves) is improved by a small margin, so the relatively sufficient 2min conduct of final choice Principle component extraction
The extraction time of test solution preparation.
6, the influence of solid-liquid ratio in sample extraction is investigated
Different extraction solid-liquid ratios (0.5g-25mL, 0.5g-50mL, 1g-25mL, 1g-50mL) are investigated to hawthorn broken wall medicine materical crude slice
The influence of extraction effect, specific step is as follows.
Hawthorn broken wall medicine materical crude slice is taken, each accurately weighed 2 parts of 0.5g and 2 part of 1g are set in stuffed conical flask, and 1 is added 70% methanol
25mL, No. 2 are added 70% methanol 50mL, No. 3 70% methanol 25mL of addition, No. 4 70% weighed weight of methanol 50mL of addition, and 1.2
Ten thousand turns of homogenate 2min take out, are cooled to room temperature, supply weightlessness with 70% methanol, shake up, again through 0.22 μm of miillpore filter after coarse filtration
Filtering, takes filtrate.
Using the chromatographic condition of above-mentioned fixation, above-mentioned 4 parts of samples are analyzed, the extraction effect of Different Extraction Method is compared.With
Sample spectrogram appearance number is more, peak is evenly distributed and a height of selection criteria of chlorogenic acid content, screens solid-liquid ratio.As a result such as Figure 15.
As can be seen from the results, the extraction effect no significant difference of 4 groups of solid-liquid ratios, main peak type are consistent.Wherein, solid-liquid ratio 1g-
The response at sample spectrogram principal component peak obtained by 25mL is slightly higher, separately goes out from the angle for saving material, Extraction solvent and protection environment
Hair, the solid-liquid ratio of final choice 1g-25mL.
7, the final preparation method of test solution
It is final to determine hawthorn broken wall medicine materical crude slice HPLC fingerprint map analyzing test solution according to above every investigation content
The preparation method comprises the following steps:
Hawthorn broken wall is taken to drink 1.0g, it is accurately weighed, it sets in stuffed conical flask, 70% methanol 25mL of addition, weighed weight,
1.2 ten thousand turns of homogenate 2min take out, are cooled to room temperature, supply weightlessness with 70% methanol, shake up, again through 0.22 μm of micropore after coarse filtration
Membrane filtration takes filtrate to obtain the final product.
The optimization of 5 chromatographic condition of embodiment
1, the selection of Detection wavelength
Full wavelength scanner is carried out to test sample, selects best detection wavelength from its 3D figure.Inspection target are as follows: under the wavelength
Analysis signal response is big, and peak information content is more, and peak is evenly distributed and baseline is steady, and each peak separating degree is preferable.
By calling the discovery of 3D spectrum analysis, at 280nm, chromatographic peak contains much information in hawthorn broken wall medicine materical crude slice, and responds
It is worth higher, wavelength of the final choice 280nm as hawthorn broken wall medicine materical crude slice fingerprint map analyzing.
2, the selection of flow phase system
Investigate following flow phase system: (1) methanol-water；(2) acetonitrile-water；(3) -0.1% phosphoric acid solution of methanol；(4) first
- 0.1% formic acid solution of alcohol；(5) -0.1% acetic acid of methanol (36% acetic acid) solution；(6) -0.1% glacial acetic acid solution of methanol；(7)
The phosphoric acid solution of acetonitrile -0.1%；(8) -0.1% formic acid solution of acetonitrile；(9) -0.1% acetic acid of acetonitrile (36% acetic acid) solution；
(10) -0.1% glacial acetic acid solution of acetonitrile, -0.1% formic acid solution of (11) acetonitrile-methanol.
Hawthorn broken wall medicine materical crude slice test solution is prepared using finally determining sample solution preparation method.
Other stationary chromatographic conditions are as follows: Shiseido CAP Cell MGIII C18(5 μm, 250mm × 4.6mm) chromatography
Column；Flow velocity 1mL/min；30 DEG C of column temperature；Detection wavelength 280nm；10 μ L of test sample sample volume.
Compare different elution systems to the separating effect of hawthorn broken wall medicine materical crude slice sample composition, with the appearance number of analysis of spectra
More, peak is evenly distributed, baseline is steady, and No. 4 peak energy are split up into selection criteria substantially.
The result shows that: binary mobile phase elution system cannot separate well 4,5, No. 6 at swarming, and use mobile phase
The peak information for the test sample spectrogram that system 11 (- 0.1% aqueous formic acid ternary mobile phase of acetonitrile-methanol) analysis obtains is more,
Baseline is more steady, and separating degree, peak shape and column are imitated preferably (see Figure 16), therefore finally determines that mobile phase is -0.1% first of acetonitrile-methanol
Acid solution.
3, gradient optimizing
Hawthorn broken wall medicine materical crude slice test solution is prepared using finally determining sample solution preparation method.
Chromatographic condition: Shiseido CAP Cell MGIII C18(5 μm, 250mm × 4.6mm) chromatographic column；Flow velocity 1mL/
min；30 DEG C of column temperature；Detection wavelength 280nm；10 μ L of test sample sample volume；10 μ L of reference substance sample volume；Using acetonitrile as mobile phase A,
0.1% aqueous formic acid is Mobile phase B, by gradient elution shown in table 8, records chromatogram.By the analysis of the above chromatographic condition for examination
Product solution, difference of the more different gradients to hawthorn broken wall medicine materical crude slice separating effect, the more, peak point with sample spectrogram appearance number
Cloth is uniform, and No. 4 peak energy are split up into selection criteria substantially.
8 eluent gradient table of table
As a result as shown in figure 17, the appearance number of each gradient is almost the same；4,5, No. 6 of gradient 1-8 elution gained spectrogram
It is poor at swarming separating degree；The baseline of gradient 11 is steady, and peak is evenly distributed, and 4,5, No. 6 at swarming good separating effect, therefore selects
Gradient 11.
4, the selection of flow velocity
Investigate test solution elution flow rate be respectively 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min,
The analytical effect of 1mL/min, 1.1mL/min, 1.2mL/min.
Hawthorn broken wall medicine materical crude slice test solution is prepared using finally determining sample solution preparation method.
Other chromatographic conditions: Shiseido CAP Cell MGIII C18(5 μm, 250mm × 4.6mm) chromatographic column；Flowing
It is mutually -0.1% aqueous formic acid of acetonitrile-methanol；It is eluted using above-mentioned gradient 11；30 DEG C of column temperature；Detection wavelength 280nm；For examination
10 μ L of product sample volume.Influence of the different elution flow rates to hawthorn broken wall medicine materical crude slice separating effect is investigated, with sample spectrogram appearance time
Moderate, principal component peak separating degree is high, be evenly distributed alternatively standard.
As a result as shown in figure 18: compared with other flow velocitys, when the flow velocity of mobile phase is 0.7mL/min, the spectrum of test sample
Figure appearance time is moderate, and peak separating degree is higher, is evenly distributed, and is selected as optimum flow rate.
5, the selection of chromatographic column column temperature
Sample analysis effect when column temperature is respectively 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C is investigated, to excellent by column temperature
Change further increases peak separating degree.
Other chromatographic conditions: Shiseido CAP Cell MGIII C18(5 μm, 250mm × 4.6mm) chromatographic column；Flowing
It is mutually -0.1% aqueous formic acid of acetonitrile-methanol；It is eluted using above-mentioned gradient 11；Detection wavelength 280nm；Flow velocity is 0.7mL/
min；10 μ L of test sample sample volume.
As a result as shown in figure 19: when column temperature is 20~40 DEG C, the separating degree difference of each main chromatographic peak is little；Binding experiment
Actual environment, 30 DEG C of final choice as final chromatographic column temperature.
7, the investigation of test solution sampling volume
Investigate the analytical effect that test solution sampling volume is respectively 5 μ L, 10 μ L, 15 μ L, 20 μ L.
Other chromatographic conditions: Shiseido CAP Cell MGIII C18(5 μm, 250mm × 4.6mm) chromatographic column；Flowing
It is mutually 0.1% aqueous formic acid of acetonitrile-methanol-；It is eluted using above-mentioned gradient 11；Flow velocity is 0.7mL/min；30 DEG C of column temperature；
Detection wavelength 280nm.Influence of the test sample difference sampling volume to hawthorn broken wall medicine materical crude slice separating effect is investigated, with sample chromatogram figure
The responsiveness height and the good alternatively standard of separating degree at middle peak.
As a result as shown in figure 20: 4 groups of sampling volume chromatogram no significant differences, final choice sampling volume are 20 μ L.
7, final chromatographic condition
According to above every investigation content, the final chromatographic condition for determining fingerprint of hawthorn as are as follows:
Chromatographic column: Shiseido CAP Cell chromatographic column (250mm × 4.6mm, 5 μm) chromatographic column；Flow velocity: 0.7mL/min；Inspection
Survey wavelength: 280nm；Column temperature: 30 DEG C；Sampling volume: 20 μ L of test solution；10 μ L of reference substance solution；Mobile phase and gradient are such as
Shown in the following table 9.
The volume fraction of each mobile phase during 9 gradient elution of table
| Time (min) | Methanol (volume fraction/%) | Acetonitrile (volume fraction/%) | 0.1% aqueous formic acid (volume fraction/%) |
| 0 | 0 | 5 | 95 |
| 10 | 2 | 8 | 90 |
| 20 | 2 | 8 | 90 |
| 30 | 2 | 11 | 87 |
| 45 | 2 | 14 | 84 |
| 70 | 2 | 28 | 70 |
| 75 | 0 | 5 | 95 |
| 85 | 0 | 5 | 95 |
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range can also make it on the basis of above description and thinking for those of ordinary skill in the art
Its various forms of variation or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should be included in the protection model of the claims in the present invention within mind and principle
Within enclosing.
Claims (10)
1. a kind of construction method of the HPLC finger-print of hawthorn broken wall medicine materical crude slice, which comprises the following steps:
S1. prepared by test solution: precision weighs hawthorn broken wall medicine materical crude slice, is 1:(25~100 by mass volume ratio) addition 50~
100% methanol, in 1.2 ten thousand~1.7 ten thousand turns of 1~5 min of homogenized after infiltration, taking-up is cooled to room temperature, with 50~100% methanol
Weightlessness is supplied, is shaken up, takes filtrate up to test solution through 0.22 μm of filtering with microporous membrane again after coarse filtration；
S2. prepared by reference substance solution: using chlorogenic acid as reference substance, methanol being added to be configured to reference substance solution；
The chromatographic condition of S3.HPLC analysis: test solution obtained by S1 is crossed into octadecylsilane chemically bonded silica chromatographic column, column temperature
20~40 DEG C；Gradient elution is carried out by mobile phase of -0.1% formic acid solution of acetonitrile-methanol；Flow velocity is 0.6~1.2 mL/min；
Detection wavelength is 280 nm；
Mobile phase acetonitrile volume fraction during gradient elution changes are as follows: 0~10 min, 5%~8%；10 min~20
Min, 8%~8%；20 min~30 min, 8%~11%；30 min~45 min, 11%~14%；45 min~70 min, 14%
~28%；70 min~75 min, 28%~5%；75 min~85 min, 5%~5%；
0.1% formic acid solution of mobile phase volume fraction during gradient elution changes are as follows: 0~10 min, 95%~90%；
10 min~20 min, 90%~90%；20 min~30 min, 90%~87%；30 min~45 min, 87%~84%；45
Min~70 min, 84%~70%；70 min~75 min, 70%~95%；75 min~85 min, 95%~95%；
S4. test solution is analyzed under the chromatographic condition of step S3 to get the HPLC finger-print of hawthorn broken wall medicine materical crude slice.
2. construction method according to claim 1, which is characterized in that the matter of hawthorn broken wall medicine materical crude slice and methanol described in step S1
Amount volume ratio is 1:25.
3. construction method according to claim 1, which is characterized in that the volume fraction of methanol described in step S1 is 70%.
4. construction method according to claim 1, which is characterized in that the condition of homogenized described in step S1 is 1.2 ten thousand
Turn, the time of processing is 2 min.
5. construction method according to claim 1, which is characterized in that reference substance solution described in step S2 passes through following
Step is prepared: precision weighs chlorogenic acid reference substance, and adding methanol to be configured to concentration is 0.5 mg/mL reference substance solution.
6. a kind of hawthorn broken wall medicine materical crude slice HPLC finger-print, which is characterized in that pass through the building of any one of claim 1 to 5
Method constructs to obtain.
7. a kind of hawthorn broken wall medicine materical crude slice standard HPLC finger-print, which is characterized in that be by any one of claim 1 to 5 institute
Construction method is stated, building obtains the HPLC finger-print of 10 batch hawthorn broken wall medicine materical crude slice samples, the HPLC finger-print that will be obtained
It imports fingerprint map analyzing software, generates finger-print common pattern to obtain the final product；The standard HPLC finger-print has 11 to share
Peak.
8. hawthorn broken wall medicine materical crude slice standard HPLC finger-print according to claim 7, which is characterized in that the standard HPLC refers to
Line map has 11 shared peaks, and using the retention time at No. 6 peaks as reference, the relative retention time at remaining 10 shared peak is respectively
0.278、0.295、0.393、0.746、0.888、1.130、1.180、1.423、1.438、1.456。
9. hawthorn broken wall medicine materical crude slice standard HPLC described in hawthorn broken wall medicine materical crude slice HPLC finger-print, claim 7 described in claim 6
Hawthorn broken wall medicine materical crude slice standard HPLC finger-print described in finger-print and/or claim 8 is in detection hawthorn broken wall prepared slice quality
In application.
10. a kind of method of quality control of hawthorn broken wall medicine materical crude slice, which comprises the steps of: take hawthorn broken wall medicine materical crude slice
Measuring samples, according to the HPLC finger-print of any one of claim 1 to the 5 construction method building measuring samples；Then with
The hawthorn broken wall medicine materical crude slice standard HPLC finger-print of claim 7 or 8 is compared；If the HPLC finger-print of measuring samples
In, 11 character pair peaks are presented, and calculate by similarity evaluation, the HPLC of measuring samples refers to
The similarity of line map and standard HPLC finger-print is not less than 0.85, is referring to peak, when remaining each peak retains relatively with No. 6 peaks
Between in the range of ± 5%, then be judged as qualified samples.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111579701A (en) * | 2020-05-19 | 2020-08-25 | 河南科高中标检测技术有限公司 | Standard fingerprint spectrum establishment method for camelina sativa seeds and method for detecting rutin content |
| CN115060812A (en) * | 2022-05-13 | 2022-09-16 | 华润三九现代中药制药有限公司 | Fingerprint spectrum of fried hawthorn medicinal preparation and construction method and application thereof |
| CN115060812B (en) * | 2022-05-13 | 2023-07-07 | 华润三九现代中药制药有限公司 | Fingerprint of fried haw medicinal preparation and construction method and application thereof |
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